RU2541806C1 - Method of obtaining preparation, which contains amorphous-crystalline salts of orotic acid - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention represents method of obtaining preparation, which contains amorphous or amorphous-crystalline salts of orotic acid, which includes thermal processing of crystalline potassium orotate and/or magnesium orotate with the following milling in milling activating device; thermal processing constitutes in thermostatting at temperature 115-125°C for 40-60 min, and as milling activating device ball epicyclical miller is applied; milling is carried out for the time, necessary for supply of specific energy 7-10 kJ/g.

EFFECT: creation of preparations based on of orotic acid salts, characterised by increased solubility, rate of solution and biological availability.

6 dwg, 4 tbl

Description

The invention relates to the field of the pharmaceutical industry and can be used to produce biologically active substances, therapeutic and prophylactic agents.

Magnesium and potassium are the main trace elements of the human body. Magnesium activates about three hundred enzymes, all enzyme systems in which ATP takes part as a substrate, which is necessary for a wide variety of energy processes during carbohydrate, protein and lipid metabolism, as well as in the synthesis of nucleic acids.

The main role of potassium in the body is to maintain the functioning of cell walls. In addition, it helps to maintain the concentration of magnesium and its physiological functions.

The advantages of potassium and magnesium orotates in comparison with other drugs - simple salts of organic acids (for example, potassium and magnesium aspartates, which are part of asparkam and panangin), lies in the fact that the pharmacological effect of the drug is determined not only by metal cations, but also by orotate anion .

Orotic acid not only takes part in magnesium metabolism, but also has an independent metabolic effect. Orotic acid is a direct precursor of pyrimidine bases - one of the constituent nucleic acids (DNA and RNA). It has been established that orotic acid is a cardioprotector: it accelerates myocardial regeneration, increases resistance to ischemia, and heart attack survival. It is orotic acid that is necessary for fixing magnesium on ATP in the cell.

A means is known that includes as an active principle an effective amount of potassium orotate and target additives in an amount of from 45 to 55% by weight of the active substance (RU 2173998). Milk sugar, potato or corn starch, edible gelatin, stearic acid were used as target additives.

Known drug Magnerot in the form of tablets containing magnesium orotate company Worwag Pharma, Germany (Vidal Handbook, Astra Pharm Service, 2006, p. 695).

The disadvantages of the known drugs are the lack of effectiveness and low rate of absorption of magnesium, which leads to the need for a longer intake of drugs and an increase in the drug load on the body.

Known pharmaceutical composition (patent RU 2225713), combining two active substances - potassium orotate and magnesium. The simultaneous use of the tablet preparations “Magnerot” and “potassium orotate” will lead to the same effect on the human body as the use of the drug “Orokamag”.

The effectiveness of the known drugs based on potassium and magnesium orotates is limited by poor solubility. As a result, relatively high doses and a long course of treatment are necessary to obtain the treatment effect.

An increase in solubility can be achieved due to co-crystallization with a well-soluble component [Dushkin A.V., Suntsova L.P., Halikov S.S. Mechanochemical technology to increase the solubility of drugs. Basic research No. 1, 2013, p. 450]. Co-crystallization is carried out by mechanochemical reaction. The result is actually a new substance with a new chemical formula: “due to the translation of drugs into their water-soluble salts (if the drug has pronounced acid-base properties), as well as due to the inclusion of drug molecules in supramolecular water-soluble formations (intermolecular complexes, micelles) with specially selected "auxiliary" substances. " The disadvantages of this method include the need for comprehensive clinical trials, the identification of possible side effects of the resulting new substance.

It is known that the biological properties of drugs depend on the crystal structure (amorphous or crystalline), the polymorphic modification of the crystal structure, as well as on the isomeric structure of the molecules. Therefore, a promising way is to change the structure of known drugs in order to increase their therapeutic effectiveness.

The dissolution rate depends on the surface area of contact of water with a solid substance, therefore, an increase in dispersion leads to an increase in the dissolution rate. Amorphization also, as a rule, leads to an increase in the rate of dissolution. In this case, the value of the solute (solubility) does not change.

A known method of obtaining a pharmaceutical composition (RU 2452480), selected as a prototype, which is carried out by joint machining of glycine enriched with gamma-crystalline modification of glycine, in an amount of 90-98% and the addition of non-toxic organic acids in an amount of 2-10%, in a vibration mill components of the composition for 6-60 minutes. The solubility of the composition increases due to changes in the pH of the stomach when using organic acids, and the dissolution rate due to dispersion of the powder and changes in the crystalline modification of glycine as a result of mechanical processing. The disadvantage of this method is that these effects on the selected components have a weak effect on the bioavailability of the active substance, which is determined by the mechanism of interaction of the molecule of this substance with the lipid layer from the gastrointestinal tract and the mechanism of interaction of the molecule of the active substance with cell membranes. Dispersion accelerates the dissolution rate of a substance, practically without changing its solubility (amount of solute), a change in crystalline modification affects the process of absorption from the gastrointestinal tract. However, both of these factors do not affect the mechanism of interaction of the molecule of the dissolved active substance with the cell membrane. This mechanism is determined by steric factors - the availability of certain functional groups and the electronic structure of the molecule. When implementing the method do not control the dose of transmitted energy during mechanical activation.

The present invention proposes to use the mechanical activation of orotates in a mixture with auxiliary substances in a planetary ball mill. Mechanoactivation leads to a change in the isomeric structure of orotate - tautomeric transformation. Three tautomers, oxo, hydroxy and dihydroxy forms are known for the orotate anion. The difference in the electronic structure of tautomeric forms is as follows.

The oxo form contains two pyrrole nitrogen atoms (-NH-) e carbonyl groups (> C = O). The hydroxy form has one pyrrole and one pyridine (-N =) nitrogen atom, one carbonyl and one hydroxyl (-C-OH) group. The dioxi form has two pyridine nitrogen atoms and two hydroxyl groups.

The pyridine nitrogen atom has an unshared pair of electrons and exhibits basic properties. The lone pair of electrons of the pyrrole nitrogen atom is part of the aromatic system and the nitrogen atom can serve as the center of acidity. The trigonal sp ² -hybridized carbon atom of the carbonyl group forms three σ-bonds lying in the same plane and p-bond with oxygen due to the non-hybridized p-orbital. Due to the difference in the electronegativity of the carbon and oxygen atoms, the n-bond between them is strongly polarized. As a result, an effective positive charge arises on the carbon atom of the carbonyl group, and a negative charge arises on the oxygen atom. The electron-deficient carbon atom becomes the center of the nucleophilic reaction. The hydroxyl group attached to the heterocycle exhibits a mesomeric effect, as a result of which hydrogen acquires mobility and the ability to dissociate in aqueous solutions. The hydroxyl group is less hydrophilic than the carbonyl group.

The difference in the electronic structure of the orotate anion leads to different solubilities in water, aqueous solutions, and lipids. The most important is the difference in the mechanisms of interaction of different tautomers with proteins of the cell membrane.

The technical result of the present invention is the creation of preparations based on salts of orotic acid, characterized by increased solubility, dissolution rate and digestibility (bioavailability).

The technical result of the present invention is the creation of preparations based on salts of orotic acid, characterized by increased solubility and dissolution rate in water, lipophilicity and bioavailability.

The technical result is achieved in a method for producing a preparation containing amorphous or amorphous crystalline salts of orotic acid with a high content of the hydroxy form (lactim isomer) of orotates, including heat treatment of crystalline potassium orotate and / or magnesium orotate, followed by grinding in a grinding activator device. Heat treatment involves thermostating at a temperature of 115-125 ° C for 40-60 minutes to remove crystalline hydrated water. A ball planetary mill is used as a grinding activator device, grinding is carried out for the time required to supply a specific energy of 7-10 kJ / g.

Processing in a grinding activator device leads to a change in the structural state of orotate anions — the tautomeric conversion of oxo to the hydroxy and dihydroxy forms. The hydroxy form has the highest biological activity due to increased lipophilicity, which causes absorption from the gastrointestinal tract, the presence of two C = C double bonds, and the mechanism of interaction of the orotate anion with carbonyl and hydrophilic groups with cell membrane proteins. Dispersion, partial or complete amorphization of the mixture as a result of machining leads to an increase in the dissolution rate of the mixture.

The substances obtained are judged by the data of emission spectroscopy, X-ray diffraction, IR, XPS spectroscopy.

According to the analysis by emission spectroscopy with inductively coupled (argon) plasma (Spectroflame spectrometer), there are no inorganic impurities in the mechanically activated potassium and magnesium orotates, the source of which can be balls and walls of grinding vessels.

Methods of X-ray diffraction, X-ray electron spectroscopy and IR spectroscopy indicate that as a result of mechanical activation there is no change in the chemical composition of the mixtures, destruction or the formation of new substances.

The invention is illustrated by drawings.

FIG. 1-2 - X-ray diffraction patterns of the initial (1) and mechanically activated (2-7 kJ / g; 3-42 kJ / g) magnesium and potassium orotates, respectively;

FIG. 3 - X-ray electron C1s and N1s spectra of potassium orotate: 1 - initial; 2 - mechanically activated (7 kJ / g);

FIG. 4 - X-ray electron C1s and N1s spectra of magnesium orotate: 1 - initial; 2 - mechanically activated (7 kJ / g);

FIG. 5-6 IR spectra of potassium and magnesium orotates, respectively: 1 - initial; 2 - mechanically activated (7 kJ / g).

A method of obtaining a preparation containing amorphous or amorphous-crystalline salts of orotic acid involves the heat treatment of crystalline potassium orotate and / or magnesium orotate, followed by grinding in a grinding activator device.

Heat treatment involves thermostating at a temperature of 115-125 ° C for 40-60 minutes to remove crystalline hydrated water. The removal of crystalline hydrate water is necessary because the tautomeric conversion of the oxo form to the hydroxy or dihydroxy form, depending on the amount of hydrated water, is either impossible or to a small extent: crystalline hydrate is associated with hydrophilic carbonyl (> C = O) groups of the orotate anion , and prevents the transition of the hydrogen atom from the - NH - group to the oxygen of the carbonyl group. In addition, heat treatment accelerates the process of tautomeric transformation. Without preliminary heat treatment, the result is achieved not after 1 hour, but after several hours (approximately 3.5 hours).

A ball planetary mill is used as a grinding activator device, grinding is carried out for the time required to supply a specific energy of 7-10 kJ / g. The supplied energy can be defined as the energy intensity of the mill (W / g) × the mass of the substance (g) × the processing time (s), for example, by the method of test objects when a substance or several substances are processed until a specific structure or new substance is formed. From thermodynamics determine how much energy is needed to spend on such a process. In our experiment, the rotation speed of the mill was 890 rpm, the processing time was 2-2.5 hours.

In FIG. Figure 1-2 shows the diffraction patterns of samples of mixtures of starting and mechanically activated orotates. It can be seen that in the initial state, the orotates have a crystalline structure (diffraction patterns 1 in Fig. 1-2). After 1 h (7 kJ / g) of mechanical activation, the structure of magnesium orotate becomes amorphous-crystalline: this is evidenced by the smearing of the reflections of the X-ray diffraction pattern (diffraction pattern 2 in Fig. 2), and after 6 h (42 kJ / g) of mechanical activation, the orotate of magnesium completely amorphizes ( diffractogram 3 in Fig. 2). The structure of potassium orotate after 1 h (7 kJ / g) of mechanical activation becomes amorphous-crystalline and practically does not change even after 6 h (42 kJ / g) of mechanical activation (diffraction patterns 2 and 3 in Fig. 1).

In FIG. Figure 3 shows the XPS spectra of potassium and magnesium orotates before and after mechanical activation (7 kJ / g). An analysis of the spectra indicates a change in the atomic structure of orotates — the conversion of the lactam form to the lactim form, as a result of which an aromatic bond system is formed in the nitrogen-containing heterocycle:

in the C1s and N1s spectra of orotates, after mechanical activation, no components appear that indicate the formation of new chemical compounds;

in the C1s and N1s spectra of mechanically activated potassium orotate, the intensity of the components of carbon and nitrogen atoms in the aromatic heterocycle increases (bond C = N); in the O1s spectrum, the intensity of the component from OH groups increased;

in the C1s spectra of mechanically activated magnesium orotate, the intensity of the component from carbon atoms in the O = C-N composition remains relatively high, and the relative contribution of the component from carbon atoms in the C-OH bond is lower than in the spectra of potassium orotate.

In the IR spectrum (Fig. 5-6) of a mechanically activated sample of potassium orotate (7 kJ / g), an absorption band appears at ~ 3390 cm ^-1 , which is responsible for the stretching vibrations of the OH group. Changes in the spectral region of 950–1100 cm ^–1 are noted, which may be due to “breathing” vibrations (NC) of the pyrimidine ring, as well as stretching vibrations of the CO group. These effects indicate the tautomeric transformation of the orotate anion with the formation of an aromatic ring.

After mechanical activation in the IR spectrum of the magnesium orotate sample (Fig. 4), the fine structure of the absorption bands of 950-1100, 1500-1800, and 2800-3700 cm ^-1 disappears, which may be a result of a decrease in the induced dipole interaction with an increase in the intermolecular distance as a result of amorphization. Changes associated with the formation of an aromatic ring in the spectrum of mechanically activated orotate magic are hardly noticeable against the background of changes associated with amorphization.

The biological response of an organism to a drug, first of all, depends on its solubility, which determines the distribution of the substance in this organism and largely determines the pharmacokinetic properties of the drug. Solubility has a significant effect on the penetration of the drug from the intestine into the blood, that is, on processes such as absorption, filtration, diffusion, etc.

In the table. Figure 1 shows the solubilities of the starting and mechanically activated orotates depending on the acidity (pH) of the medium. Acidity is selected in accordance with the acidity of the environment of various parts of the stomach and intestines.

The solubility of mechanically activated potassium and magnesium orotates is higher than the initial ones, and it depends on the acidity of the medium (pH). The reason for the increased solubility of orotates is the formation of an aromatic heterocycle, which contributes to an increase in solubility in polar solvents, which include water.

The rate of dissolution in water (pH 6.5) of mechanically activated potassium orotate is higher than the initial: for example, in the first 20 minutes, the dissolution rate is 4.9 mg / l · min and 6.6 mg / l · min for the initial and mechanically activated (3 h) samples, respectively.

The dissolution rate of mechanically activated magnesium orotate in water (pH 6.5) is the same as the initial one.

The increase in the dissolution rate of orotates is due to amorphization of the samples as a result of mechanical activation.

Table 1 Solubility of orotates of potassium and magnesium Substance, dose of mechanical energy, kJ / g Concentration g / 100 g H ₂ O pH 2 pH 4 pH 6.5 pH 8.6 Potassium orotate 0 0.1 0.25 0.04 0.18 7 0.1 0.26 0.06 0.24 21 0.12 0.25 0.08 0.30 42 0.15 0.28 0.08 0.30 Magnesium Orotate 0 0.14 0.23 0.075 0.20 7 0.16 0.23 0.08 0.20 21 0.18 0.26 0.08 0.23 42 0.21 0.29 0.08 0.24

No interaction of auxiliary substances (starch, lactose, microcellulose) with potassium and magnesium orotates during mechanical activation is observed. These substances are introduced to facilitate the tabletting of mechanically activated drugs. The introduction of auxiliary substances before the mechanical activation process ensures their uniform mixing with orotates.

A change in the physicochemical properties of mechanically activated orotates compared with the initial ones indicates an increase in bioavailability as a result of mechanical activation. This characteristic was investigated by microelectrophoresis. The studied epithelial cells were placed in aqueous solutions of potassium and magnesium orotates and, using the device described, for example, in patent RU 2168176, created multi-vector, symmetric, alternating electric fields. Under the microscope, the reactions of cells to the effects of electric fields were determined by the reciprocating movements of cells or their nuclei, cytolemma.

The results of a study of the biological activity of potassium and magnesium orotates by microelectrophoresis were studied on blood cells and buccal epithelial cells (Tables 2 and 3).

table 2 Indicators of bioelectric activity of buccal epithelial cells Substance, dose of mechanical energy, kJ / g Indicators of bioelectric activity of buccal epithelial cells The proportion of active cells% Vibration amplitude the cores cytolemma cells Potassium orotate 0 52 ± 3.2 0 0.25 ± 0.1 2.1 ± 0.3 7 84 ± 2.8 8 ± 0.3 2.5 ± 0.3 3.2 ± 0.4 21 72 ± 2.6 2 ± 0.2 2.0 ± 0.4 2.2 ± 0.3 42 36 ± 3.6 0 1.7 ± 0.3 0 Magnesium Orotate 0 28 ± 3.6 0 1 ± 0.2 1 ± 0.3 7 100 ± 12.0 6 ± 0.4 1.9 ± 0.3 2.7 ± 0.3 21 100 ± 2.0 1 ± 0.2 1.2 ± 0.2 2.1 ± 0.3 42 100 ± 2.1 1 ± 0.2 0.6 ± 0.1 1.5 ± 0.2

The increase in the biological activity of potassium and magnesium orotates after mechanical activation is due to the following points:

- dispersing and forming an amorphous (for magnesium orotate) and amorphous-crystalline (for potassium orotate) structure, leading to an increase in the rate of dissolution in water;

- the formation of an aromatic heterocycle of the diroxoform of potassium orotate, which helps to increase solubility in polar solvents (water) and improve absorption from the gastrointestinal tract;

- the formation of OH groups instead of C = O as a result of the formation of the hydroxo form of magnesium orotate and the dihydrox form of potassium orotate, which leads to an increase in the hydrophobicity of orotate molecules, i.e. increased lipophilicity contributing to the improvement of the absorption of mechanically activated orotates by the body.

The use of mechanically activated potassium and magnesium orotates affects the intravital properties of cells: plasmolemma and cell nuclei are activated, lymphocyte activation is observed.

Table 3 Indicators of bioelectric activity of blood cells Substance, dose of mechanical energy, kJ / g Indicators of bioelectric activity of blood cells The proportion of active red blood cells,% Vibration amplitude Lymphocytes Potassium orotate 0 63 ± 3.1 5.5 ± 1.3 No active lymphocytes 7 88 ± 2.7 14.5 ± 2.1 There are active lymphocytes 21 88 ± 2.7 11 ± 1.9 There are active lymphocytes 42 93 ± 2.5 11.5 ± 2.2 There are active lymphocytes Magnesium Orotate 0 83 ± 2.6 7 ± 1.7 Lymphocytes are not active 7 96 ± 2.5 16 ± 3.2 There are active lymphocytes 21 92 ± 2.8 12 ± 3.0 There are active lymphocytes 42 83 ± 3.2 10 ± 2.8 Lymphocytes are active

An increase in biological activity is observed for orotates mechanically activated at a dose of mechanical energy in the range of 7-10 kJ / g.

Example

A membrane of the gastrointestinal tract was simulated on a pair of immiscible liquids water-1-octanol. Almost 2 times more potassium orotate and 1.7 times more magnesium orotate passed from aqueous solutions of the hydroxy form to octanol. This indicates a greater number of hydroxy-forms of potassium and magnesium orotates, which have passed from the gastrointestinal tract to the body. The conclusion is confirmed by studies in laboratory rats.

For studies in laboratory rats using the hypomagnesemia model (GME), the most active hydroxy form and one of the less active hydroxy forms of magnesium orotate were selected based on microelectrophoresis data. Hypomagnesemia was achieved by administering to rats for 14 days an intraperitoneal diuretic furosemide (Furosemidi 1%) at a dose of 30 mg / kg. In the state of GME, the magnesium content in the blood decreased almost 2 times compared with the initial state of the rats. When taking the oxo form, the aftereffect of furesemide continued and the magnesium content continued to decrease. When taking the hydroxy form, a positive trend was observed - the restoration of the concentration of magnesium in the blood serum.

Table 4 Comparative analysis of the magnesium content in the blood serum of healthy laboratory rats after hypomagnesemia and after taking magnesium orotate Control group Comparison Group (GME) Reception within 6 days Hydroxy form Oxo form Magnesium 1.75 ± 0.08 0.902 ± 10.18 1.12 ± 0.10 0.855 ± 10.05

Claims (1)

A method of obtaining a preparation containing amorphous or amorphous-crystalline salts of orotic acid, including heat treatment of crystalline potassium orotate and / or magnesium orotate, followed by grinding in a grinding activator device, heat treatment consists in thermostating at a temperature of 115-125 ° C for 40-60 minutes, and as a grinding activator device, a ball planetary mill is used, grinding is carried out for the time required to supply a specific energy of 7-10 kJ / g.

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